The process of drug discovery is presently undergoing a fundamental revolution as the era of functional genomics comes of age. The term “functional genomics” applies to an approach utilising bioinformatics tools to ascribe function to protein sequences of interest. Such tools are becoming increasingly necessary as the speed of generation of sequence data is rapidly outpacing the ability of research laboratories to assign functions to these protein sequences.
As bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
Various institutions and commercial organisations are examining sequence data as they become available and significant discoveries are being made on an on-going basis. However, there remains a continuing need to identify and characterise further genes and the polypeptides that they encode, as targets for research and for drug discovery.
Alternative pre-mRNA splicing is a major cellular process by which functionally diverse proteins can be generated from the primary transcript of a single gene, often in tissue specific patterns.
Experimentally, splice variants are identified by the fortuitous isolation and subsequent sequencing of variant mRNAs. However, this experimental approach has not been exhaustively completed for the human transcriptome (since this would require systematic isolation and sequencing of all mRNAs from all human tissues under all possible environmental conditions) and due to this experimental limitation there remains a large number of splice variants which have yet to be identified.
We have used proprietary bioinformatic approaches to perform a purposeful, directed search for the existence of splice variants of the human growth hormone gene. By this method the limited data set of experimentally known splice variants can be extended to a much larger set of predicted splice variants.
Endocrine Hormones
Hormones regulate a wide variety of physiological functions encompassing intermediary metabolism, growth and cell differentiation. They have two fundamental mechanisms of action, depending on their physical chemical characteristics. The lipophilic steroid hormones and thyroid hormones are hydrophobic and act primarily intracellularly, modulating gene transcription, whereas the peptide hormones such as adrenaline and melatonin are hydrophilic and act at the cell membrane, triggering a cascade of signal transduction events leading to intracellular regulatory effects (Lodish et al. (1995) Molecular Cell Biology, Scientific American Books Inc., New York, N.Y., pp. 856-864).
Hormones are produced in specialised cells of the endocrine glands and reach their target cells by way of the blood circulation. The steroid hormones are derived from cholesterol by a series of enzymatic reactions that take place in the cytosol and in mitochondria of primarily cells of the adrenal cortex, ovary, and testis. In some cases the steroid hormone must be subjected to modification in the target tissue, either to be activated or to produce a more active derivative. Most of the peptide hormones are synthesized in the form of precursor proteins (prohormones) and are stored in the endocrine cell. Before being released into the circulation, the prohormone is cleaved to the active hormone. Several hormones (primarily steroid hormones and thyroid hormones) are transported in the circulation while bound to specific binding proteins. These proteins serve as hormone depots, releasing the hormone when needed and also protecting it from rapid inactivation.
Because of the central nature of hormones in the general physiology of H. sapiens, the dys-regulation of hormonal function has been shown to play a role in many disease processes, including, but not limited to oncology (Sommer S. and Fuqua S. A. (2001) Semin Cancer Biol. October; 11(5):339-52, Bartucci M., Morelli C., Mauro L, Ando S., and Surmacz E. (2001) Cancer Res. September 15; 61(18):6747-54, Oosthuizen G. M., Joubert G., and du Toit R. S. (2001) S. Afr. Med. J. July; 91(7):576-79, Nickerson T., Chang F., Lorimer D., Smeekens S. P., Sawyers C. L., and Pollak M. (2001) Cancer Res. August 15; 61(16):6276-80) cardiovascular disease (Liu Y., Ding J., Bush T. L., Longenecker J. C., Nieto F. J., Golden S. H., and Szklo M. (2001) Am. J. Epidemiol. September 15; 154(6):489-94), metabolic diseases (Flyvbjerg A. (2001) Growth Horm. IGF Res. June; 11 Suppl. A:S115-9, Diamond T., Levy S., Smith A., Day P. and Manoharan A. (2001) Intern. Med. J. July; 31(5):272-8, Toprak S., Yonem A., Cakir B., Guler S., Azal O., Ozata M., and Corakci A. (2001) Horm. Res.; 55(2):65-70), inflammation (McEvoy A. N., Bresnihan B., FitzGerald O., and Murphy E. P. (2001) Arthritis Rheum. August; 44(8):1761-7, Lipsett P. A. (2001) Crit. Care Med. August; 29(8):1642-4) and CNS related diseases (Bowen R. L. (2001) JAMA. August 15; 286(7):790-1).
Growth Hormone Family
Growth hormone is a member of a family of polypeptide hormones that share structural similarities and biological activities and are produced in the pituitary glands of all vertebrates and the placentae of some mammals. Family members include pituitary prolactin, placental lactogens (also called chorionic somatomammotropins in humans [hCS]), prolactin-related proteins in ruminants and rodents, proliferins in mice, and somatolactin in fish.
The genes that encode most members of the GH family comprise five exons and four introns and appear to have arisen by duplication of a single ancestral gene prior to the appearance of the vertebrates. Splicing and processing variants have been described for several members of the family.
The human GH-related gene family located on chromosome 17q22-24 consists of a gene cluster of highly sequence-conserved genes and a single prolactin gene on chromosome 6 (Owerbach D. et al. Science 1981). The gene cluster includes five structural genes, two GH and three CS genes, whose expression is tissue specific: hGH-N (N=normal), hGH-V (V=variant), human chorionic somatomammotropin hormone-like (hCS-L), human chorionic somatomammotropin A and B (hCCS-A and hCS-B) (Misra-Press, A et al. JBC 1994; Boguszewski C. et al. JBC 1998).
The GH-related family of proteins has shared structural similarities since their tertiary structure form four ∝-helices, also known as a four antiparallel helix bundle. The ∝-helices are tightly packed and arranged in an antiparallel up-up-down-down orientation, with two long loops linking the parallel pairs.
The hGHM hCS gene family is important in the regulation of maternal and fetal metabolism and the growth and development of the fetus. During pregnancy, pituitary GH (hGH-N) expression in the mother is suppressed; and hGH-V, a GH variant expressed by the placenta, becomes the predominant GH in the mother. hCS, which is the product of the hCS-A and hCS-B genes, is secreted into both the maternal and fetal circulations after the sixth week of pregnancy. hGH-V and hCS act in concert in the mother to stimulate insulin-like growth factor (IGF) production and modulate intermediary metabolism, resulting in an increase in the availability of glucose and amino acids to the fetus. In the fetus, hCS acts via lactogenic receptors and possibly a unique CS receptor to modulate embryonic development, regulate intermediary metabolism and stimulate the production of IGFs, insulin, adrenocortical hormones and pulmonary surfactant. hGH-N, which is expressed by the fetal pituitary, has little or no physiological actions in the fetus until late in pregnancy due to the lack of functional GH receptors on fetal tissues. hGH-V, which is also a potent somatogenic hormone, is not released into the fetus. Taken together, studies of the hGH/hCS gene family during pregnancy reveal a complex interaction of the hormones with one another and with other growth factors. Additional investigations are necessary to clarify the relative roles of the family members in the regulation of fetal growth and development and the factors that modulate the expression of the genes.” (Handwerger S. & Freemark M. J., Pediatr. Endocrinol. Metab. 2000 April; 13(4):343-56).
Human growth hormone, also known as somatotropin, is a protein hormone of about 190 amino acids that is synthesized and secreted by cells called somatotrophs in the anterior pituitary. It is a major participant in control of several complex physiologic processes, including growth and metabolism. Growth hormone is also of considerable interest as a drug used in both humans and animals.
Growth hormone has two distinct types of effects. Direct effects are the result of growth hormone binding its receptor on target cells. Fat cells (adipocytes), for example, have growth hormone receptors, and growth hormone stimulates them to break down triglyceride and suppresses their ability to take up and accumulate circulating lipids. Indirect effects are mediated primarily by insulin-like growth factor-1 (IGF-1). The major role of growth hormone in stimulating body growth is to stimulate the liver and other tissues to secrete IGF-1. A majority of the growth promoting effects of growth hormone is actually due to IGF-1 acting on its target cells. For example, IGF-1 stimulates proliferation of chondrocytes (cartilage cells), resulting in bone growth. Growth hormone also has important effects on protein, lipid and carbohydrate metabolism. In some cases, a direct effect of growth hormone has been clearly demonstrated, in others, IGF-1 is thought to be the critical mediator, and some cases it appears that both direct and indirect effects are at play.
In addition to its complex effects on growth, states of both growth hormone deficiency and excess provide very visible testaments to the role of this hormone in normal physiology. Such disorders can reflect lesions in either the hypothalamus, the pituitary or in target cells. A deficiency state can result not only from a deficiency in production of the hormone, but in the target cell's response to the hormone.
Clinically, deficiency in growth hormone or receptor defects are as growth retardation or dwarfism. The manifestation of growth hormone deficiency depends upon the age of onset of the disorder and can result from either heritable or acquired disease.
The effect of excessive secretion of growth hormone is also very dependent on the age of onset and is seen as two distinctive disorders. Giantism is the result of excessive growth hormone secretion that begins in young children or adolescents. It is a very rare disorder, usually resulting from a tumour of somatotropes.
Acromegaly results from excessive secretion of growth hormone in adults. The onset of this disorder is typically insideous. Clinically, an overgrowth of bone and connective tissue leads to a change in appearance that might be described as having “coarse features”. The excessive growth hormone and IGF-1 also lead to metabolic derangements, including glucose intolerance.
Growth hormone purified from human cadaver pituitaries has long been used to treat children with severe growth retardation. More recently, the availability of recombinant growth hormone has lead to several other applications to human and animal populations. For example, human growth hormone is commonly used to treat children of pathologically short stature. The role of growth hormone in normal aging remains poorly understood, but some of the cosmetic symptoms of aging appear to be amenable to growth hormone therapy. Growth hormone is currently approved and marketed for enhancing milk production in dairy cattle; another application of growth hormone in animal agriculture is treatment of growing pigs with porcine growth hormone. Such treatment has been demonstrated to significantly stimulate muscle growth and reduce deposition of fat.
As growth hormone plays such a key role in cellular processes, the study of this moiety and its method of regulation are of key interest. The identification of splice variants of this hormone would be of great scientific importance.